It has long been suggested that the import of nuclease colicins requires protein processing; however it had never been formally demonstrated. Here we show that two RNase colicins, E3 and D, which appropriate two different translocation machineries to cross the outer membrane (BtuB/Tol and FepA/TonB, respectively), undergo a processing step inside the cell that is essential to their killing action. We have detected the presence of the C-terminal catalytic domains of these colicins in the cytoplasm of target bacteria. The same processed forms were identified in both colicin-sensitive cells and in cells immune to colicin because of the expression of the cognate immunity protein. We demonstrate that the inner membrane protease FtsH is necessary for the processing of colicins D and E3 during their import. We also show that the signal peptidase LepB interacts directly with the central domain of colicin D in vitro and that it is a specific but not a catalytic requirement for in vivo processing of colicin D. The interaction of colicin D with LepB may ensure a stable association with the inner membrane that in turn allows the colicin recognition by FtsH. We have also shown that the outer membrane protease OmpT is responsible for alternative and distinct endoproteolytic cleavages of colicins D and E3 in vitro, presumably reflecting its known role in the bacterial defense against antimicrobial peptides. Even though the OmpT-catalyzed in vitro cleavage also liberates the catalytic domain from colicins D and E3, it is not involved in the processing of nuclease colicins during their import into the cytoplasm.Colicins are antibacterial toxins of Escherichia coli that are released into the extracellular medium in response to environmental stress conditions. Colicin D is an RNase that cleaves the anticodon loop of all four isoaccepting tRNA Arg (1). Colicin E3 cleaves 16 S ribosomal RNA (2). Both colicins provoke cell death by inactivating the protein biosynthetic machinery. Colicin producer cells are protected against both endogenous and exogenous toxin molecules by the constitutive expression of a cognate immunity (Imm) 3 protein, which forms a tight heterodimer complex with the nuclease domain of the colicin (3, 4).Colicin E3, like most colicins, has structurally identifiable N-terminal, central, and C-terminal domains. The first two domains are required for translocation and receptor binding, and they "hijack" certain functions of the target cell (i.e. the BtuB receptor and the Tol system) during colicin import. The C-terminal domain carries the cell-killing RNase function (5, 6). The colicin D protein has an unusual tripartite organization. The N-terminal domain is required for both the binding of the colicin to the high affinity, iron siderophore receptor FepA and for its subsequent translocation across the outer membrane. The 280-residue central domain is essential for uptake (and thus for cell killing), and it is also involved in the formation of the colicin D-ImmD protein complex (7). The passage of colicin D through th...

Many biological scenarios generate "dirty" DNA 3′-PO 4 ends that cannot be sealed by classic DNA ligases or extended by DNA polymerases. The noncanonical ligase RtcB can "cap" these ends via a unique chemical mechanism entailing transfer of GMP from a covalent RtcB-GMP intermediate to a DNA 3′-PO 4 to form DNA 3′ pp 5′ G. Here, we show that capping protects DNA 3′ ends from resection by Escherichia coli exonucleases I and III and from end-healing by T4 polynucleotide 3′ phosphatase. By contrast, the cap is an effective primer for DNA synthesis. E. coli DNA polymerase I and Mycobacterium DinB1 extend the DNAppG primer to form an alkali-labile DNApp(rG)pDNA product. The addition of dNTP depends on pairing of the cap guanine with an opposing cytosine in the template strand. Aprataxin, an enzyme implicated in repair of A 5′ pp 5′ DNA ends formed during abortive ligation by classic ligases, is highly effective as a DNA 3′ decapping enzyme, converting DNAppG to DNA 3′ p and GMP. We conclude that the biochemical impact of DNA capping is to prevent resection and healing of a 3′-PO 4 end, while permitting DNA synthesis, at the price of embedding a ribonucleotide and a pyrophosphate linkage in the repaired strand. Aprataxin affords a means to counter the impact of DNA capping.DNA repair | 3′ exonuclease T he synthesis and repair of DNA are driven by enzymatic reactions at 3′ ends. The terminal 3′-OH is the nucleophile that primes 3′-5′ phosphodiester bond formation by DNA polymerases and DNA ligases. To contend with the many situations in biology when incision of DNA generates a 3′-PO 4 (a so-called "dirty" end, which cannot be extended by polymerases or sealed by classic ligases), nature has evolved an assortment of endhealing enzymes that remove the 3′-PO 4 and/or resect 3′ nucleotides (1). We recently described a different fate for DNA 3′-PO 4 ends at the hands of the unconventional ligase RtcB (2, 3), which adds a guanylate "cap" to form DNA 3′ pp 5′ G (4). DNA capping by RtcB is highly efficient, whether at the 3′-PO 4 ends of ssDNAs or at a nick in duplex DNA (4). DNA 3′ capping has tantalizing implications. For analogy, one need only consider the multifaceted role of the 5′ cap structure, m 7 GpppN-, in RNA metabolism (5). Here, we address the consequences of DNA 3′ capping for the reactions of exemplary exonucleases, phosphatases, and polymerases at 3′ ends. In addition, we illuminate aprataxin as a DNA 3′ decapping enzyme. Results and DiscussionDNA Capping Confers Resistance to End Resection by 3′ Exonucleases and 3′ Phosphatase. Escherichia coli exonucleases I and III are DNA repair enzymes that hydrolyze 3′-OH DNA ends in a stepwise fashion to liberate 5′ deoxyribonucleoside monophosphate (dNMP) mononucleotide products. ExoI acts processively on ssDNA with a 3′-OH end but has minimal activity at a 3′-PO 4 end (6, 7). By contrast, ExoIII distributively resects 3′-OH and 3′-PO 4 DNA ends, the latter by virtue of its intrinsic DNA 3′ phosphatase activity (7-9). Here, we gauged the effect of DNA 3′ capping on end r...

Escherichia coli DNA ligase (EcoLigA) repairs 3′-OH/5′-PO4 nicks in duplex DNA via reaction of LigA with NAD+ to form a covalent LigA-(lysyl-Nζ)–AMP intermediate (step 1); transfer of AMP to the nick 5′-PO4 to form an AppDNA intermediate (step 2); and attack of the nick 3′-OH on AppDNA to form a 3′-5′ phosphodiester (step 3). A distinctive feature of EcoLigA is its stimulation by ammonium ion. Here we used rapid mix-quench methods to analyze the kinetic mechanism of single-turnover nick sealing by EcoLigA–AMP. For substrates with correctly base-paired 3′-OH/5′-PO4 nicks, kstep2 was fast (6.8–27 s−1) and similar to kstep3 (8.3–42 s−1). Absent ammonium, kstep2 and kstep3 were 48-fold and 16-fold slower, respectively. EcoLigA was exquisitely sensitive to 3′-OH base mispairs and 3′ N:abasic lesions, which elicited 1000- to >20000-fold decrements in kstep2. The exception was the non-canonical 3′ A:oxoG configuration, which EcoLigA accepted as correctly paired for rapid sealing. These results underscore: (i) how EcoLigA requires proper positioning of the nick 3′ nucleoside for catalysis of 5′ adenylylation; and (ii) EcoLigA's potential to embed mutations during the repair of oxidative damage. EcoLigA was relatively tolerant of 5′-phosphate base mispairs and 5′ N:abasic lesions.

′ -OH nick termini, k step2 was fast (9.5 to 17.9 sec −1 ) and similar in magnitude to k step3 (7.9 to 32 sec −1 ). Rnl2 fidelity was enforced mainly at the level of step 2 catalysis, whereby 3 ′ -OH base mispairs and oxoguanine, oxoadenine, or abasic lesions opposite the nick 3 ′ -OH elicited severe decrements in the rate of 5 ′ -adenylylation and relatively modest slowing of the rate of phosphodiester synthesis. The exception was the noncanonical A:oxoG base pair, which Rnl2 accepted as a correctly paired end for rapid sealing. These results underscore (1) how Rnl2 requires proper positioning of the 3 ′ -terminal ribonucleoside at the nick for optimal 5 ′ -adenylylation and (2) the potential for nick-sealing ligases to embed mutations during the repair of oxidative damage.

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